The hygiene hypothesis proposes that deficiencies in exposure to microbes, due to modern public health practices, underlies the epidemic of allergic diseases presently experienced in industrialized countries. Toll like receptor ligands (TLR) have been shown to play a critical role in innate immune activation by microbes. Studies from this and other laboratories further suggest that TLRs and their ligands may form an important molecular bridge between microbes, immune development, and the prevention of the allergic march, providing a mechanistic framework for understanding how hygiene effects the development of atopy. A protective role for environmental TLR stimulation against the allergic phenotype is strongly suggested by our recent observation that mice with global deficiency in TLR ligand responsiveness (MyD88 ko mice) have intrinsically Th2 biased immunoreactivity, and increased bronchial hyperresponsiveness, compared to wild type mice. Consistent with these observations, we and others have shown that several of the TLR ligands protect against the allergic phenotype. The overriding goal of this program project is to understand the molecular and cellular basis of the hygiene hypothesis. The first Project will explore genetic considerations. The third project will explore innate immunity, and the fourth project will explore the adaptive immunity, in the context of TLR signaling and the hygiene hypothesis. In project two we will characterize the role of environmental exposure to TLR ligands in immune maturation and propensity towards allergic manifestations. Specifically, we will pursue the following specific aims: (SA-1) to determine the effects of pharmacological doses of TLR ligands on APC, T cell, and B cell maturation and differentiation (SA-2) to characterize the role of physiologic TLR stimulation in neonatal immune development, and (SA-3) to evaluate the effects of deficient TLR stimulation and TLR based interventions on the allergic phenotype. To achieve these goals, we will utilize purified and quality controlled TLR ligands and Myd88 and TLR deficient mice. By addressing the specific aims of the second project we anticipate a better understanding of relationship between environmental exposure to TLR ligands, the hygiene hypothesis, and the pathogenesis of allergic diseases.